Enhanced generation of cytotoxic T lymphocytes by IL-21 mediated FoxP3 suppression

ABSTRACT

A method of carrying out adoptive immunotherapy by administering a subject an antigen-specific cytotoxic T lymphocytes (CTL) preparation in a treatment-effective amount is described. In the method, the CTL preparation is preferably administered as a preparation of an in vitro antigen-stimulated and expanded primate CTL population, the CTL population: (i) depleted of FoxP3+ T lymphocytes prior to antigen stimulation; (ii) antigen-stimulated in vitro in the presence of interleukin-21; or (iii) both depleted of FoxP3+ T lymphocytes prior to antigen stimulation and then antigen-stimulated in vitro in the presence of interleukin-21. Methods of preparing such compositions, and compositions useful for carrying out the adoptive immunotherapy, are also described.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/677,035, filed Mar. 8, 2010, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2008/011088, filed Sep. 25, 2008, which claims priority to U.S.Provisional Application No. 60/977,150, filed Oct. 3, 2007, each ofwhich is incorporated herein by reference in its entirety.

This invention was made with government support under grant numberCA083636 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention concerns methods and compositions for carrying outadoptive immunotherapy.

BACKGROUND OF THE INVENTION

Adoptive cellular therapy of cancer involves ex vivo generation ofautologous antigen specific T cells, followed by in vitro expansion andinfusion into patients in the hope that transferred T cells will trafficto and eradicate tumor^(1,2). Recent studies have demonstrated tumorregression and measurable clinical benefit in patients who are otherwiserefractory to conventional therapy^(3,4). One major obstacle to adoptivetherapy has been the feasibility of isolating tumor-reactive T cells. Tcells recognizing tumor-associated antigens, which are often normal selfproteins, exist at very low frequency and exhibit predominantly lowtarget avidity^(5,6) due to central and peripheral tolerance mechanisms.Moreover, such potentially autoreactive T cells may be suppressed bycirculating regulatory cells (T_(reg)), that can lead to impairedproliferative response to antigenic stimulation.

Regulatory T cells (T_(reg)) have been shown to play a critical role incontrolling immunologic tolerance to self-antigens as well as presentinga major barrier to the development of effective anti-tumor immunity inanimal models⁷⁻⁹. In animal models, tumor antigen specific CD8 cellsfailed to undergo normal functional maturation in the presence ofT_(reg) cells and were rendered incapable of destroying specific tumortargets¹⁰. Conversely, depletion of regulatory T cells controlled thegrowth of melanoma in most mice and promoted long-lasting CD8⁺T-cell-dependent protective immunity¹¹, possibly through the recruitmentof high-avidity antigen specific CTL¹². Recent evidence of elevatedT_(reg) cells in the peripheral blood of patients with cancer¹³⁻¹⁶ andthe finding that increased prevalence of tumor-associated T_(reg) insitu as a predictor for reduced survival¹⁷ suggest the importance ofregulatory control of the endogenous anti-tumor immune response. Thesuppressive mechanisms at play in vivo¹⁸, may also limit the capacity togenerate antigen-specific T cells in vitro.

Expression of the forkhead transcription factor, Foxp3, has been linkedto the regulatory phenotype. Although Foxp3 is a reliable marker ofnaturally occurring T_(reg), as an intracellular protein, it cannot beused as a practical method of sorting for viable T_(reg) cells. Instead,it has been shown that CD4+ cells constitutively expressing CD25^(hi),are also FoxP3+. We and others have demonstrated that depletion of CD25+cells in vitro can lead to enhanced generation of CD4+ T cellsrecognizing tumor-associated self antigens¹⁹ presumably by eliminatingthe inhibitory influence of CD25+ T regs in the PBMC responderpopulation.

IL-21 belongs to the family of gamma-chain receptor cytokines thatincludes IL-2, IL-7, and IL-15-cytokines that all deliver theirintracellular signal through the shared gamma-chain receptor andinfluence T cell activation and differentiation²⁰⁻²². Recently, wedemonstrated that in vitro exposure to IL-21 (in contrast to othergamma-chain receptor cytokines) can lead to the generation of selfantigen-specific CTL in increased numbers and with enhanced avidity andfunction²³. In light of work demonstrating that barriers to optimal Tcell development may involve a regulatory component, we postulate thatIL-21 may influence the regulatory control of cellular responses totumor self proteins in vitro.

SUMMARY OF THE INVENTION

A first aspect of the present invention is, in a method of carrying outadoptive immunotherapy in a primate subject in need thereof byadministering the subject an antigen-specific cytotoxic T lymphocytes(CTL) preparation in a treatment-effective amount, the improvementcomprising: administering as the CTL preparation a preparationconsisting essentially of an in vitro antigen-stimulated and expandedprimate CTL population, the CTL population: (z) depleted of FoxP3+ Tlymphocytes (or depleted of CD25+ cells) prior to antigen stimulation;(ii) antigen-stimulated in vitro in the presence of interleukin-21; or(iii) both depleted of FoxP3+ T lymphocytes (or depleted of CD25+ cells)prior to antigen stimulation and then antigen-stimulated in vitro in thepresence of interleukin-21.

A second aspect of the present invention is a pharmaceutical formulationcomprising or consisting essentially of an in vitro expanded primatecytotoxic T lymphocyte (CTL) population, the CTL population depleted ofFoxP3+ T lymphocytes (or depleted of CD25+ cells) prior to antigenstimulation and the CTL population antigen-stimulated in vitro in thepresence of interleukin-21.

A further aspect of the present invention is the use of a formulation asdescribed herein for the preparation of a medicament for treatingcancer, or for treating an infectious disease, in a primate subject inneed thereof.

A further aspect of the invention is a method of making a cytotoxic Tlymphocyte (CTL) preparation useful for adoptive immunotherapy; themethod comprising the steps of: (a) optionally (but in some embodimentspreferably) separating a lymphocyte subpopulation depleted of FoxP3+cells (or depleted of CD25+ cells) therefrom from a first lymphocytepopulation collected from a donor; (b) enriching for antigen-specificCTL in the subpopulation in vitro in a culture medium optionally (but insome embodiments preferably) containing interleukin-21; and then (c)collecting cells from the culture medium to produce the CTL preparation.

In some embodiments of the invention, the FoxP3+ cells (or CD25+ cells)are depleted in the CTL preparation at least 2, 5, 10, 50 100-fold, andthe number of antigen specific CD8+ CTL are enriched in the CTLpreparation at least 5, 10, 50, 100 or 200-fold, as compared to thatseen in the same CTL preparation not subjected to the separating step(b) and where interleukin-21 is not included in the culture medium inthe expanding step (c).

A further aspect of the invention is an in vitro expanded primatecytotoxic T lymphocyte (CTL) preparation produced by the processesdescribed herein.

A further aspect of the invention is, in a method of carrying outadoptive immunotherapy in a primate subject in need thereof byadministering the subject an antigen-specific cytotoxic T lymphocytes(CTL) preparation in a treatment-effective amount, the improvementcomprising: administering as the CTL preparation a preparationconsisting essentially of an in vitro antigen-stimulated and expandedprimate CTL population, the CTL population depleted of FoxP3+ Tlymphocytes prior to antigen stimulation in vitro; while concurrentlyadministering the subject interleukin-21 in an amount effective topromote the expansion of the antigen-specific cytotoxic T lymphocytes inthe subject. (ii) antigen-stimulated in vitro in the presence ofinterleukin-21; or (iii) both depleted of FoxP3+ T lymphocytes prior toantigen stimulation and then antigen-stimulated in vitro in the presenceof interleukin-21.

A further aspect of the invention is, in a method of carrying outadoptive immunotherapy in a primate subject in need thereof byadministering the subject an antigen-specific cytotoxic T lymphocytes(CTL) preparation in a treatment-effective amount, the improvementcomprising: administering the subject a CD25 depletion agent (e.g.,daclizumab, diphtheria toxin conjugated to IL-2) in an amount effectiveto deplete the CTL population of FoxP3+ T lymphocytes in the subject;and concurrently administering the subject interleukin-21 in an amounteffective to promote the expansion of the antigen-specific cytotoxic Tlymphocytes in the subject.

A further aspect of the invention is the use of interleukin-21 for thepreparation of a medicament for promoting the expansion ofantigen-specific cytotoxic T lymphocytes (CTL) in a subject receivingadoptive immunotherapy by administration of the CTLs.

A further aspect of the invention is the use of a CD25 depletion agent(e.g., denileukin diftitox or a pharmaceutically acceptable saltthereof; e.g., Ontak®) for the preparation of a medicament for depletinga CTL population of FoxP3+ T lymphocytes in a subject receiving adoptiveimmunotherapy by administration of the CTLs.

The foregoing and other objects and aspects of the present invention areexplained in greater detail in the drawings herein and the specificationbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Combined use of IL-21 and CD25 depletion leads to significantlyenhanced generation of antigen-specific CTL. Unmanipulated orCD25-depleted PBMC from HLA-A2+ patients with melanoma were stimulatedin vitro with autologous mature dendritic cells pulsed with the MART-1,M26 peptide as described in ‘Materials and Method’. Where indicated,IL-21 (30 ng/ml) was added at culture initiation. A. After two cycles ofstimulation 5×10⁵ Cells from each experiment group were harvested andstained with 20 μg/ml of peptide/MHC tetramer (PE, vertical axis) andFITC-conjugated CD4, a vital dye (DAPI) was also added to exclude deadcells. Data are expressed as percentage of tetramer positive cells amonggated lymphocytes on day 18 after 2^(nd) stimulation. B. The absolutenumber in millions of tetramer+ cells corresponding to each experimentalculture from a representative melanoma patient depicted in A., and thefold increase in absolute numbers of CD25 depletion and/or IL-21 treatedto untreated cultures (Control). Representative results from threeseparate experiments are presented.

FIG. 2. Combined use of Il-21 and CD25 depletion enhances generation ofWT-1 and NY-ESO-1-specific CTL responses. Unmanipulated or CD25-depletedPBMC from an HLA-A2+ patient with a WT-1+ ovarian cancer (FIG. 2 A,above) and a patient with melanoma (NY-ESO seropositive) were stimulatedas described in FIG. 1. The increase in absolute number of WT-1 orNY-ESO-1-specific CTL as well as the fold increase in numbers comparedto control cultures are shown in FIG. 2B.

FIG. 3. IL-21 is unique among γ-chain receptor cytokines in its abilityto induce expansion of antigen-specific CTL. Unmanipulated orCD25-depleted PBMC from HLA-A2+ patients with melanoma were stimulatedin vitro with autologous mature dendritic cells pulsed with the MART-1,M26 peptide as described in ‘Materials and Method’. Optimalconcentrations of IL-2 (12.5 U/ml), IL-7 (10 ng/ml), IL-15 (30 U/ml),and IL-21 (30 ng/ml) were added to individual cultures at time ofstimulation on Day 1. 5×10⁵ cells from each experiment group wereharvested on Day 7 and stained with 20 μg/ml of peptide/MHC tetramer-PEand PerCP-conjugated CD8. Data are expressed as percentage of tetramerpositive cells among gated lymphocytes.

FIG. 4. IL-21-treated cultures enrich for a population of CD28^(hi)Ag-specific memory CTL. Cells were collected 7 days after 2^(nd)stimulation with MART-1 peptide-pulsed autologous DCs and stained forMART-1-tetramer and simultaneously with either CD28, CCR7 and CD45RAunder the conditions described: PBMC (untreated control culture),PBMC+IL-21 (addition of IL-21 at 30 ng/ml during the first stimulation),CD25 depl (CD25-depleted PBMC used as a source of T cells) andCD25depl+IL-21 (combined CD25 depletion and IL-21 treatment). Theseresults are representative of cultures from three donors.

FIG. 5. The frequency of CD4+Foxp3+ T cells is significantly decreasedby IL-21 and CD25 treatment in stimulation culture. MART-1-specific Tcell stimulation cultures were established using unmanipulated PBMC(control), IL-21-treated, CD25-depleted and CD25 depleted/IL-21-treatedPBMC. Three weeks following initial in vitro stimulation, 5×10⁵ cellsfrom each experimental culture were stained with FITC-conjugated CD4,followed by intracellular staining for Foxp3 protein. Data are expressedas percentage of CD4+Foxp3 positive cells among gated lymphocytes.

FIG. 6. IL-21 partially reverses T_(reg)-mediated inhibition of CD8 Tcell proliferation. A) Sorted T_(reg) cells (5×10⁴/well) were culturedwith IL-2 (12.5 U/ml), IL-7 (10 ng/ml), IL-15 (30 ng/ml) or IL-21 (30ng/ml) in the presence or absence of anti-CD3 and autologous dendriticcells; B) Sorted CD8+CD25-effector T cells (5×10⁴/well) were culturedwith Treg cells at ratio of 1:1 (CD8+:Treg) in the presence of anti-CD3and autologous dendritic cells. IL-21 (30 ng/ml) was added as indicated.

Proliferation was measured by ³H thymidine incorporation pulsed on day 3and harvested 16˜20 hours later. The results of CPM were calculated fromtriplicate wells with standard error of the mean. The p value wasobtained by applying paired sample t test to evaluate the influence ofT_(reg) on the CD8 proliferation with and without IL-21.

The present invention is explained in greater detail below. Thedisclosures of all United States patent references cited herein areincorporated by reference herein in their entirety.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

“T cells” or “T lymphocytes” as used herein may be from any mammalian,preferably primate, species, including monkeys, dogs, and humans. Insome embodiments the T cells are allogenic (from the same species butdifferent donor) as the recipient subject; in some embodiments the Tcells are autologous (the donor and the recipient are the same); in someembodiments the T cells are syngeneic (the donor and the recipients aredifferent but are identical twins).

Cytotoxic T lymphocyte (CTL) as used herein refers to a T lymphocytethat expresses CD8 on the surface thereof (i.e., a CD8⁺ T cell). CTLsadministered to patients or subjects are, in general, “antigen-specific”in that they specifically bind to a tumor-associated antigen and areable to specifically recognize and lyse cells of that tumor, orspecifically bind to a microbe-associated antigen and are able tospecifically recognize and lyse that microbe.

“Enriched” and “depleted” as used herein to describe amounts of celltypes in a mixture refers to the subjecting of the mixture of the cellsto a process or step which results in an increase in the number of the“enriched” type and a decrease in the number of the “depleted” cells.Thus, depending upon the source of the original population of cellssubjected to the enriching process, a mixture or composition may contain60, 70, 80, 90, 85, or 99 percent or more (in number or count) of the“enriched” cells and 40, 30, 20, 10, 5 or 1 percent or less (in numberor count) of the “depleted” cells.

“Interleukin-21” or “IL-21” (also previously referred to as Zalpha11Ligand) is known. The term IL-21 as used herein is intended to includenatural IL-21 (e.g., mammalian, primate or particularly human IL-21),and recombinant IL-21, as well as active fragments and analogs thereof.Natural and recombinant IL-21 and active fragments and analogs thereofare all known and described in, for example, U.S. Pat. No. 6,686,187 toNovak et al. (ZymoGenetics); U.S. Pat. No. 7,250,274 to Chan et al.(ZymoGenetics); US Patent Application 2007/0178063 to Kindsvogel et al.(ZymoGenetics); US Patent Application 2007/0071717 to Weiner et al.(University of Iowa); US Patent Application 2006/0159655 to Collins etal. (Wyeth); and US Patent Application 2005/0124044 to Cunningham et al.(Johnson & Johnson).

“Treat” as used herein refers to any type of treatment that imparts abenefit to a patient afflicted with a disease, including improvement inthe condition of the patient (e.g., in one or more symptoms), delay inthe progression of the disease, etc.

“Pharmaceutically acceptable” as used herein means that the compound orcomposition is suitable for administration to a subject to achieve thetreatments described herein, without unduly deleterious side effects inlight of the severity of the disease and necessity of the treatment.

“Concurrently” means sufficiently close in time to produce a combinedeffect (that is, concurrently may be simultaneously, or it may be two ormore events occurring within a short time period before or after eachother).

I. Antigen Stimulation and In Vitro Expansion.

Antigen stimulation is the process of enriching for a population ofantigen-specific T cells by stimulating with antigen or antigen fragment(peptide) in vitro and expansion is the method use to expand (in thecase of REM—non-specifically, but it can also be expanded specificallyusing antigen or antigen fragments), the T cells used for adoptivetherapy. Expansion typically utilizes allogeneic irradiated feedercells; Antigen stimulation typically does not; howeverantigen-stimulation does require the antigen or antigen fragment to bepresented by autologous simulator cells.

In this invention a lymphokine, particularly IL-21, is used duringantigen stimulation. Optionally, a lymphokine such as IL-21 may also beused during expansion to suppress the outgrowth of FoxP3+/Treg cellsduring the expansion process.

T lymphocytes can be collected from a suitable donor in accordance withknown techniques and stimulated, enriched or depleted by knowntechniques such as affinity binding to antibodies such as flow cytometryand/or affinity binding. After enrichment and/or depletion steps, invitro expansion of the desired T lymphocytes can be carried out inaccordance with known techniques (including but not limited to thosedescribed in U.S. Pat. No. 6,040,177 to Riddell et al.), or variationsthereof that will be apparent to those skilled in the art.

Depletion of FoxP3+ cells can be carried out by any suitable means, suchas by depletion of CD25⁺ cell, or any other physical means of removingFoxP3+ in vitro, such as depletion of cells bearing other Treg surfacemarkers, including but not limited to GITR, positive selection withCD127-pos cells for stimulation (Tregs are often CD127-neg) Moreparticularly, depletion of CD25⁺ cells from an initial population may becarried out by any suitable technique, such as by contacting the cellsto a solid support (e.g. beads) having anti-CD25 antibodies immobilizedthereon, so that cells expressing CD25 are bound to the solid support,and then separating the remaining cell subpopulation from the solidsupport.

The cells are preferably expanded in vitro following the in vitroantigen-stimulation (or enrichment) step. Expansion in vitro may becarried out by any suitable technique. For example, the desired T cellpopulation or subpopulation may be expanded by adding an initial Tlymphocyte population to a culture medium in vitro, and then adding tothe culture medium feeder cells, such as non-dividing peripheral bloodmononuclear cells (PBMC), (e.g., such that the resulting population ofcells contains at least about 5, 10, 20, or 40 or more PBMC feeder cellsfor each T lymphocyte in the initial population to be expanded); andincubating the culture (e.g. for a time sufficient to expand the numbersof T cells). The order of additional of the T cells and feeder cells tothe culture media can be reversed if desired. The culture can typicallybe incubated under conditions of temperature and the like that aresuitable for the growth of T lymphocytes. For the growth of human Tlymphocytes, for example, the temperature will generally be at leastabout 25 degrees Celsius, preferably at least about 30 degrees, morepreferably about 37 degrees.

Interleukin-21 may be included in or added to the culture medium in anysuitable amount, typically at least 1, 2 or 5 nanograms per milliliter,up to 200, 500 or 1000 nanograms per milliliter, or more. The amount canbe adjusted and optimized for particular types of interleukin-21 andparticular culture techniques by simply monitoring the amount of FoxP3⁺cells in the expanded product, and/or monitoring the amount ofantigen-specific CTLs in the expanded product, and adjusting the amountas indicated or desired.

The T lymphocytes expanded are typically cytotoxic T lymphocytes (CTL)that are specific for an antigen present on a human tumor or a pathogen.

The non-dividing feeder cells can comprise gamma-irradiated PBMC feedercells. In some embodiments, the PBMC are irradiated with gamma rays inthe range of about 3000 to 3600 rads.

Optionally, the expansion method may further comprise the step of addingnon-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells.LCL can be irradiated with gamma rays in the range of about 6000 to10,000 rads. The LCL feeder cells may be provided in any suitableamount, such as a ratio of LCL feeder cells to initial T lymphocytes ofat least about 10:1.

Optionally, the expansion method may further comprise the step of addinganti-CD3 monoclonal antibody to the culture medium (e.g., at aconcentration of at least about 0.5 ng/ml). Optionally, the expansionmethod may further comprise the step of adding IL-2 and/or IL-15 to theculture medium (e.g., wherein the concentration of IL-2 is at leastabout 10 units/ml).

In some embodiments it may be desired to introduce functional genes intothe T cells to be used in immunotherapy in accordance with the presentinvention. For example, the introduced gene or genes may improve theefficacy of therapy by promoting the viability and/or function oftransferred T cells; or they may provide a genetic marker to permitselection and/or evaluation of in vivo survival or migration; or theymay incorporate functions that improve the safety of immunotherapy, forexample, by making the cell susceptible to negative selection in vivo asdescribed by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); andRiddell et al., Human Gene Therapy 3:319-338 (1992); see also thepublications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al.,describing the use of bifunctional selectable fusion genes derived fromfusing a dominant positive selectable marker with a negative selectablemarker. This can be carried out in accordance with known techniques(see, e.g., U.S. Pat. No. 6,040,177 to Riddell et al. at columns 14-17)or variations thereof that will be apparent to those skilled in the artbased upon the present disclosure.

Various infection techniques have been developed which utilizerecombinant infectious virus particles for gene delivery. Thisrepresents a currently preferred approach to the transduction of Tlymphocytes of the present invention. The viral vectors which have beenused in this way include virus vectors derived from simian virus 40,adenoviruses, adeno-associated virus (AAV), and retroviruses. Thus, genetransfer and expression methods are numerous but essentially function tointroduce and express genetic material in mammalian cells. Several ofthe above techniques have been used to transduce hematopoietic orlymphoid cells, including calcium phosphate transfection, protoplastfusion, electroporation, and infection with recombinant adenovirus,adeno-associated virus and retrovirus vectors. Primary T lymphocyteshave been successfully transduced by electroporation and by retroviralinfection.

Retroviral vectors provide a highly efficient method for gene transferinto eukaryotic cells. Moreover, retroviral integration takes place in acontrolled fashion and results in the stable integration of one or a fewcopies of the new genetic information per cell.

It is contemplated that overexpression of a stimulatory factor (forexample, a lymphokine or a cytokine) may be toxic to the treatedindividual. Therefore, it is within the scope of the invention toinclude gene segments that cause the T cells of the invention to besusceptible to negative selection in vivo. By “negative selection” ismeant that the infused cell can be eliminated as a result of a change inthe in vivo condition of the individual. The negative selectablephenotype may result from the insertion of a gene that conferssensitivity to an administered agent, for example, a compound. Negativeselectable genes are known in the art, and include, inter alia thefollowing: the Herpes simplex virus type I thymidine kinase (HSV-I TK)gene (Wigler et al., Cell 11:223, 1977) which confers ganciclovirsensitivity; the cellular hypoxanthine phosphribosyltransferase(HPRT)gene, the cellular adenine phosphoribosyltransferase (APRT) gene,bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci.USA. 89:33 (1992)).

In some embodiments it may be useful to include in the T cells apositive marker that enables the selection of cells of the negativeselectable phenotype in vitro. The positive selectable marker may be agene which, upon being introduced into the host cell expresses adominant phenotype permitting positive selection of cells carrying thegene. Genes of this type are known in the art, and include, inter alia,hygromycin-B phosphotransferase gene (hph) which confers resistance tohygromycin B, the aminoglycoside phosphotransferase gene (neo or aph)from Tn5 which codes for resistance to the antibiotic G418, thedihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA),and the multi-drug resistance (MDR) gene.

Preferably, the positive selectable marker and the negative selectableelement are linked such that loss of the negative selectable elementnecessarily also is accompanied by loss of the positive selectablemarker. Even more preferably, the positive and negative selectablemarkers are fused so that loss of one obligatorily leads to loss of theother. An example of a fused polynucleotide that yields as an expressionproduct a polypeptide that confers both the desired positive andnegative selection features described above is a hygromycinphosphotransferase thymidine kinase fusion gene (HyTK). Expression ofthis gene yields a polypeptide that confers hygromycin B resistance forpositive selection in vitro, and ganciclovir sensitivity for negativeselection in vivo. See Lupton S. D., et al, Mol. and Cell. Biology11:3374-3378, 1991. In addition, in preferred embodiments, thepolynucleotides of the invention encoding the chimeric receptors are inretroviral vectors containing the fused gene, particularly those thatconfer hygromycin B resistance for positive selection in vitro, andganciclovir sensitivity for negative selection in vivo, for example theHyTK retroviral vector described in Lupton, S. D. et al. (1991), supra.See also the publications of PCT/US91/08442 and PCT/US94/05601, by S. D.Lupton, describing the use of bifunctional selectable fusion genesderived from fusing a dominant positive selectable markers with negativeselectable markers.

Preferred positive selectable markers are derived from genes selectedfrom the group consisting of hph, neo, and gpt, and preferred negativeselectable markers are derived from genes selected from the groupconsisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt.Especially preferred markers are bifunctional selectable fusion geneswherein the positive selectable marker is derived from hph or neo, andthe negative selectable marker is derived from cytosine deaminase or aTK gene.

A variety of methods can be employed for transducing T lymphocytes, asis well known in the art. For example, retroviral transductions can becarried out as follows: on day 1 after stimulation using REM asdescribed herein, provide the cells with 20-30 units/ml IL-2; on day 3,replace one half of the medium with retroviral supernatant preparedaccording to standard methods and then supplement the cultures with 5ug/ml polybrene and 20-30 units/ml IL-2; on day 4, wash the cells andplace them in fresh culture medium supplemented with 20-30 units/mlIL-2; on day 5, repeat the exposure to retrovirus; on day 6, place thecells in selective medium (containing, e.g., an antibiotic correspondingto an antibiotic resistance gene provided in the retroviral vector)supplemented with 30 units/ml IL-2; on day 13, separate viable cellsfrom dead cells using Ficoll Hypaque density gradient separation andthen subclone the viable cells.

II. Compositions and Methods.

Subjects that can be treated by the present invention are, in general,human and other primate subjects, such as monkeys and apes forveterinary medicine purposes. The subjects can be male or female and canbe any suitable age, including infant, juvenile, adolescent, adult, andgeriatric subjects.

Subjects that can be treated include subjects afflicted with cancer,including but not limited to colon, lung, liver, breast, prostate,ovarian, skin (including melanoma), bone, and brain cancer, etc. In someembodiments the tumor associated antigens are known, such as melanoma,breast cancer, squamous cell carcinoma, colon cancer, leukemia, myeloma,prostate cancer, etc. (in these embodiments memory T cells can beisolated or engineered by introducing the T cell receptor genes). Inother embodiments the tumor associated proteins can be targeted withgenetically modified T cells expressing an engineered immunoreceptor.Examples include but are not limited to B cell lymphoma, breast cancer,prostate cancer, and leukemia.

Subjects that can be treated also include subjects afflicted with, or atrisk of developing, an infectious disease, including but not limited toviral, retroviral, bacterial, and protozoal infections, etc.

Subjects that can be treated include immunodeficient patients afflictedwith a viral infection, including but not limited to Cytomegalovirus(CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus infectionsin transplant patients, etc.

Cells prepared as described above can be utilized in methods andcompositions for adoptive immunotherapy in accordance with knowntechniques, or variations thereof that will be apparent to those skilledin the art based on the instant disclosure. See, e.g., US PatentApplication Publication No. 2003/0170238 to Gruenberg et al; see alsoU.S. Pat. No. 4,690,915 to Rosenberg.

In some embodiments, the cells are formulated by first harvesting themfrom their culture medium, and then washing and concentrating the cellsin a medium and container system suitable for administration (a“pharmaceutically acceptable” carrier) in a treatment-effective amount.Suitable infusion medium can be any isotonic medium formulation,typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter),but also 5% dextrose in water or Ringer's lactate can be utilized. Theinfusion medium can be supplemented with human serum albumen.

A treatment-effective amount of cells in the composition is at least10⁹, typically greater than 10⁹, at least 10¹⁰ cells, and generally morethan 10¹⁰. The number of cells will depend upon the ultimate use forwhich the composition is intended as will the type of cells includedtherein. For example, if cells that are specific for a particularantigen are desired, then the population will contain greater than 70%,generally greater than 80%, 85% and 90-95% of such cells. For usesprovided herein, the cells are generally in a volume of a liter or less,can be 500 mls or less, even 250 mls or 100 mls or less. Hence thedensity of the desired cells is typically greater than 10⁶ cells/ml andgenerally is greater than 10⁷ cells/ml, generally 10⁸ cells/ml orgreater. The clinically relevant number of immune cells can beapportioned into multiple infusions that cumulatively equal or exceed10⁹, 10¹⁰ or 10¹¹ cells.

In some embodiments, the lymphocytes of the invention may be used toconfer immunity to individuals. By “immunity” is meant a lessening ofone or more physical symptoms associated with a response to infection bya pathogen, or to a tumor, to which the lymphocyte response is directed.The amount of cells administered is usually in the range present innormal individuals with immunity to the pathogen. Thus, the cells areusually administered by infusion, with each infusion in a range of atleast 10⁶ to 10¹⁰ cells/m², preferably in the range of at least 10⁷ to10⁹ cells/m². The clones may be administered by a single infusion, or bymultiple infusions over a range of time. However, since differentindividuals are expected to vary in responsiveness, the type and amountof cells infused, as well as the number of infusions and the time rangeover which multiple infusions are given are determined by the attendingphysician, and can be determined by routine examination. The generationof sufficient levels of T lymphocytes (including cytotoxic T lymphocytesand/or helper T lymphocytes) is readily achievable using the rapidexpansion method of the present invention, as exemplified herein. See,e.g., U.S. Pat. No. 6,040,177 to Riddell et al. at column 17.

III. Active Agents for In Vivo Administration.

In some embodiments of the invention, subjects being treated areadministered a CD25+ cell depleting agent. Depletion of such cells canbe effected using any of a variety pharmaceutically acceptable agents,including small molecules and antibodies (e.g., monoclonal antibodies,preferably, humanized monoclonal antibodies). Antibodies that bindspecifically to the alpha subunit (p55 alpha, CD25, or Tac subunit) ofthe human high-affinity interleukin-2 receptor that is expressed on thesurface of activated lymphocytes are particularly preferred, ZENAPAX(daclizumab) being a specific example. Alternatively, diphtheria toxinconjugated to IL-2, such as ONTAK, can be used (e.g., in humans) toeffect transient depletion of T regulatory cells. See, e.g., B Haynes etal., US Patent Application Publication No. 2006/0165687 at paragraph 39therein. Such compounds are sometimes referred to as “active agents” or“active compounds” herein.

In some embodiments of the invention, subjects being treated areadministered IL-21 as an active agent. Such agents (also sometimesreferred to as “active agents” or “active compounds” herein) are knownas described above.

The active compounds disclosed herein can, as noted above, be preparedin the form of their pharmaceutically acceptable salts. Pharmaceuticallyacceptable salts are salts that retain the desired biological activityof the parent compound and do not impart undesired toxicologicaleffects. Examples of such salts are (a) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; and saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; (b)salts formed from elemental anions such as chlorine, bromine, andiodine, and (c) salts derived from bases, such as ammonium salts, alkalimetal salts such as those of sodium and potassium, alkaline earth metalsalts such as those of calcium and magnesium, and salts with organicbases such as dicyclohexylamine and N-methyl-D-glucamine.

The active compounds described above may be formulated foradministration in a pharmaceutical carrier in accordance with knowntechniques. See, e.g., Remington, The Science And Practice of Pharmacy(9^(th) Ed. 1995). In the manufacture of a pharmaceutical formulationaccording to the invention, the active compound (including thephysiologically acceptable salts thereof) is typically admixed with,inter alia, an acceptable carrier. The carrier must, of course, beacceptable in the sense of being compatible with any other ingredientsin the formulation and must not be deleterious to the patient. Thecarrier may be a solid or a liquid, or both, and is preferablyformulated with the compound as a unit-dose formulation, for example, atablet, which may contain from 0.01 or 0.5% to 95% or 99% by weight ofthe active compound. One or more active compounds may be incorporated inthe formulations of the invention, which may be prepared by any of thewell known techniques of pharmacy comprising admixing the components,optionally including one or more accessory ingredients.

The formulations of the invention include those suitable for oral,rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g.,subcutaneous, intramuscular, intradermal, or intravenous), topical(i.e., both skin and mucosal surfaces, including airway surfaces) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated and on the nature of the particular active compound which isbeing used.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above). In general, the formulations of the invention are preparedby uniformly and intimately admixing the active compound with a liquidor finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet may be prepared bycompressing or molding a powder or granules containing the activecompound, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing, in a suitable machine, thecompound in a free-flowing form, such as a powder or granules optionallymixed with a binder, lubricant, inert diluent, and/or surfaceactive/dispersing agent(s). Molded tablets may be made by molding, in asuitable machine, the powdered compound moistened with an inert liquidbinder.

Formulations suitable for buccal (sub-lingual) administration includelozenges comprising the active compound in a flavoured base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound(s), which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The formulations may bepresented in unit\dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising an activecompound(s), or a salt thereof, in a unit dosage form in a sealedcontainer. The compound or salt is provided in the form of alyophilizate which is capable of being reconstituted with a suitablepharmaceutically acceptable carrier to form a liquid compositionsuitable for injection thereof into a subject. The unit dosage formtypically comprises from about 10 mg to about 10 grams of the compoundor salt. When the compound or salt is substantially water-insoluble, asufficient amount of emulsifying agent which is physiologicallyacceptable may be employed in sufficient quantity to emulsify thecompound or salt in an aqueous carrier. One such useful emulsifyingagent is phosphatidyl choline.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable for transdermal administration may also be delivered byiontophoresis (see, for example, Pharmaceutical Research 3 (6):318(1986)) and typically take the form of an optionally buffered aqueoussolution of the active compound. Suitable formulations comprise citrateor bis\tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2Mactive ingredient.

In addition to active compound(s), the pharmaceutical compositions maycontain other additives, such as pH-adjusting additives. In particular,useful pH-adjusting agents include acids, such as hydrochloric acid,bases or buffers, such as sodium lactate, sodium acetate, sodiumphosphate, sodium citrate, sodium borate, or sodium gluconate. Further,the compositions may contain microbial preservatives. Useful microbialpreservatives include methylparaben, propylparaben, and benzyl alcohol.The microbial preservative is typically employed when the formulation isplaced in a vial designed for multidose use. Of course, as indicated,the pharmaceutical compositions of the present invention may belyophilized using techniques well known in the art.

Dosages of active agents may be determined in accordance with knowntechniques. For example, the dosage of a diptheria toxin-interleukin-2fusion such as ONTAK may be from 1, 2 or 5 mcg/kg/day up to about 20, 40or 50 mcg/kg/day, typically administered intravenously (e.g., for one,two, three, four or five consecutive days or more), with eachadministration preferably delivered as an infusion over time (e.g., from10 to 20 minutes) rather than as a bolus injection. In an additionalexample, the dosage of an antibody such as ZENAPAX may be from 0.1, or0.5 mg/kg up to 2, 5 or 10 mg/kg. Dosage will of course vary dependingupon the particular active agent and formulation, as well as the age andcondition of the subject, as is known in the art.

The present invention is illustrated further in the examples set forthbelow.

EXPERIMENTAL

A. Materials and Methods

Depletion of CD25⁺ T_(reg) cells. Depletion of CD25⁺ T cells wasperformed using the CliniMACS CD25 MicroBeads (Miltenyi Biotech)according to manufacture's instruction. In brief, the leukapheresisproduct was washed and resuspended in PBS/EDTA (2 mM, invitrogen)supplemented with 0.5% human serum albumin (Life Technologies,Gaithersburg, Md.). Appropriate amount of CD25 MicroBeads was added andincubated for 15 minutes at room temperature with gentle mixing everyfive minutes. Cells were washed, resuspended, and applied to theAutoMACS instrument with the CD25 depletion program selected. Uponcompletion of the depletion, an aliquot of cell fraction was stained forCD25 following FACScan analysis to check the efficiency of CD25MicroBeads depletion.

Antibody plus peptide-MHC tetramer staining of T cells. APC labeledM26-MHC-Tetramer was produced in the immune monitoring lab at FredHutchinson Cancer Center based on previously described protocols²⁴. Forsample analysis, 0.5×10⁶ cells in 25 μl of 2% FCS/PBS were first stainedwith peptide tetramer-APC (final concentration of 20 μg MHC/ml) for onehour at room temperature, followed by anti-CD8-FITC or PerCP (BD,PharMingen, San Diego, Calif.) staining for 20 min at 4° C. to analyzeantigen specific T cell population. In some experiments, measure forphenotypes was carried out by anti-CD28-APC (BD, PharMingen, San Diego,Calif.) or anti-CD28-FITC (Caltag Lab, Burlingame, Calif.), anti-CCR7-PEand anti-CD45RO or anti-CD45RA-FITC (BD, PharMingen, San Diego, Calif.)staining. After washing with PBS, cells were resuspended in PBScontaining 2% FBS and DAPI was added. Data were acquired using aFACScalibur flow cytometer and CellQuest (BD) and analyzed using FlowJosoftware (Tree Star, San Carlos, Calif.).

In vitro generation of human antigen-specific CD8+ T cells. Donor bloodwas typed by the HLA Typing Lab at the Puget Sound Blood Center(Seattle, Wash.). HLA-A2+ donors were used in the study. MART-1 M26−,NY-ESO-1 NY157− and WT-1 WT126− peptide specific T cells were generatedin a manner similar to that previously described^(3,23). DC weregenerated by exposing adherent PBMC to IL-4 (500 U/ml, R&D) and GM-CSF(800 U/ml, Amgen) in AIM-V medium (Life Technologies) followed bymaturation using IL-1β at 2 ng/ml, IL-6 at 1000 U/ml, TNF-α at 10 ng/ml(R&D Systems, Minneapolis, Minn.) and PGE-2 at 1 μg/ml (Sigma) for anadditional 2 days. The mature DC population contained more than 90%CD83+DCs on day 8 as determined by FACS analysis.

Mature DCs were harvested and pulsed with 40 μg/ml of synthesizedpeptides at 2×10⁶ cell/ml in the presence of 3 μg/ml of β2 microglobulin(Scripps Lab, San Diego, Calif., USA) in PBS with 1% human serum albumin(Life Technologies, Gaithersburg, Md.) for 4 hrs at room temperature.After washing three times with sterile PBS (Life Technologies), DCs wereco-cultivated with responder T cells (unmanipulated or CD25-depleted) at5×10⁵ cells/well in 6 well plate in CTL medium, consisting of RPMI 1640(Gibco, Carlsbad, Calif.), 25 mM HEPES, 4 mM L-glutamine, 50 μM2-mercaptoethanol (Gibco), penicillin (50 U/ml), streptomycin (50 mg/ml)(Life Technologies, Gaithersburg, Md.), and 10% pooled human serum fromnormal donors. Cytokine, IL-21 (30 ng/ml) was added to the experimentalwells required after the culture initiated. In some experiments, IL-2(10 U/ml), IL-7 (10 ng/ml), or IL-15 (10 ng/ml) were added to comparethe effect of IL-21. These concentrations of IL-2, IL-7, IL-15 and IL-21were previously demonstrated to represent optimal dosing; higherconcentrations led to no benefit or a detrimental effect in thegeneration of antigen-specific CTL²³ (and data not shown). For thesecond stimulation, irradiated autologous PBMCs pulsed with antigenicpeptide were used. A total of 5 donors were evaluated, three of them forMART-1 specific responses and two others patients (ovarian cancer andNY-ESO seropositive melanoma donor) for WT-1 and NY-ESO-1 responses,respectively). Unless otherwise specified, results were obtained at 7days after the 2^(nd) in vitro stimulation.

Antibody plus peptide-MHC tetramer staining of T cells. APC labeledpeptide-MHC-Tetramers were produced in the Immune Monitoring Lab at FredHutchinson Cancer Center using established protocols²⁴. For sampleanalysis, 0.5×10⁶ cells in were first stained with peptide tetramer-APC(20 μg MHC/ml) for one hour at room temperature, followed byanti-CD8-FITC or PerCP (BD, PharMingen, San Diego, Calif.) staining for20 min at 4° C. After PBS wash, cells were resuspended and DAPI addedimmediately prior to flow cytometric analysis (FACScalibur flowcytometer and CellQuest (BD)) and analyzed using FlowJo software (TreeStar, San Carlos, Calif.).

Intracellular Detection of Foxp3 Protein by FACS Analysis. Intracellularstaining for Foxp3 protein used PE-conjugated anti-human Foxp3 stainingset (clone PCH101, eBioscience) according to the manufacturer'sinstructions. Briefly, 10⁶ cells were stained with FITC-conjugatedanti-CD4 antibody (BD Pharmingen) first for 20 min on ice, washed, thenre-suspended in Fixation/Permeabilization buffer and incubated for 60min at 4° C.

Immune suppression assay in vitro. Suppression assay was carried outbased on the published protocol²⁵ with minor modifications. CD4+CD25⁻and CD8+CD25− T cells were (5×10⁴ cells/well) were co-cultured withT_(reg) cells (2.5 or 5×10⁴ cells/well) with 100 ng/mL anti-CD3 antibody(OKT3) in the presence of irradiated (3500 Rad) autologous dendriticcells (0.5˜1×10⁴ cells/well) in a 96-well flat-bottom plate.Proliferation was assessed by [³H]thymidine (1 μCi [0.037 MBq] per well)incorporation.

B. Results

The combination of CD25 depletion and IL-21 markedly augments theexpansion of antigen-specific CD8+ T cells following in vitrostimulation. MART-1 specific CTL was generated according to methodsdescribed above under the following conditions: no manipulation (Controlcultures), IL-21 treatment alone, CD25 depletion alone and IL-21treatment with CD25-depleted responder PBMCs. IL-21 was used at a doseof 30 ng/ml, previously shown to be optimal for generating antigenspecific T cells²⁶. Concentrations of 10 ng/ml or less had no effect andinhibition was observed when IL-21 concentration was used at 100 ng/mlor greater.

Exposure to IL-21 added once during the first in vitro stimulation ledto a marked enhancement in the frequency of MART-1 specific CTL thatcould be generated (FIG. 1A: 1.83 vs. 0.07%), confirming our previousfindings. CD25 depletion alone led to a 10-fold increase in thefrequency of antigen specific CD8+ T cells compared with controlcultures (0.73 vs. 0.07%). A striking >450-fold increase in thefrequency of antigen specific CD8+ T cells was observed through thecombined use of CD25 depletion and IL-21 treatment (31.6 vs. 0.07%).Data from three additional patients demonstrated a 160 to >300-foldincrease in the absolute numbers of antigen-specific CTL that could begenerated with combined CD25 depletion and IL-21 treatment (FIG. 1B:1.45 vs. 244.94, 1.68 vs. 462.65, 0.45 vs. 141.0). Antigen specific Tcells generated by CD25 depletion plus IL-21 demonstrated robust killingactivity against antigen-expressing tumor target (>50% lysis at an E:Tof 10:1) and IFN-gamma secretion upon recognition of specific peptideantigen in vitro assay (Data not shown).

For these initial studies, we used a canonical tumor antigenicepitope—M26 peptide of the melanoma-associated self antigen, MART-1. Toevaluate the generality of this finding, we examined CTL responses totwo other tumor-associated antigens, WT-1 and NY-ESO-1 inHLA-A*0201+patient donors. Representative results are shown forresponses generated from the PBMC of a patient donor with WT-1+ ovariantumor and the PBMC of an NY-ESO-1 seropositive patient donor withmelanoma (FIG. 2) Although the endogenous precursor frequency of CTLspecific for WT-1 (a self antigen that is prevalent at low levels amongnormal tissues) is very low and the ability to enrich for this rarepopulation of WT-1-specific CTL somewhat elusive (0.005% after two invitro stimulations), the positive influence of CD25 depletion and IL-21exposure are similar to that seen for MART-1-specific CTL responses. A8-20 fold increase in the numbers and frequency of WT-1-specific CTL isobserved with either IL-21 treatment or CD25 depletion alone compared tocontrol, but more than 250-fold greater when cultures receive both CD25depletion and IL-21 treatment. Similarly, for NY-ESO-1 specificresponses, a relative 10-fold increase with either treatment alone,increases to 100-fold with combined CD25 depletion and IL-21 exposure.

IL-21 exposure leads to optimal expansion of antigen-specific CTLfollowing CD25 depletion. Among the γ-chain receptor cytokines,including IL-2, IL-7 and IL-15, IL-21 provided the greatest effect onantigen-specific CTL expansion following CD25 depletion (FIG. 3). Whenusing untreated PBMC, IL-21 elicited a 10-fold or greater increase inantigen-specific CTL compared with no cytokine or IL-2, IL-7 orIL-15-treated cultures in congruence with our previous results.²³ Whenusing CD25-depleted PBMC, IL-21-exposed cultures yielded a 20-foldgreater increase in antigen-specific CTL frequency compared to otherCD25-depleted cultures.

IL-21 exposure enriches for a population of CD28+CCR7−CD8+Memory CTL.The surface phenotype of antigen-specific CTL generated in culturesunder the four conditions (control, IL-21 treatment, CD25 depletion, andcombined IL-21 treatment and CD25 depletion) was evaluated by stainingfor CD28, CCR7, CD45RA expression (FIG. 4). A unique memory phenotype,CD28+, CCR7−, was observed only among the IL-21 exposed cultures(PBMC+IL-21 and CD25depl+IL-2). Results were obtained 7 days after the2^(nd) in vitro stimulation. This phenotype was stable for more than 4weeks when maintained with IL-2 and IL-7 and without further exposure toIL-21. These findings are consistent with previous studies we havepublished demonstrating upregulation of CD28 expression andhelper-independence among antigen-specific CTL generated in IL-21treated cultures.²³

Exposure of CTL culture to IL-21 leads to a decrease in the frequency ofFoxp3⁺ T cells. FoxP3+ cells were identified by intracellular stainingfollowing CD25 depletion and/or IL-21 treatment (FIG. 5). CD25 depletionalone led to a measurable decrease in the fraction of FoxP3+ cells inculture. IL-21 treatment alone led to a 10-fold decrease in the fractionof FoxP3+ cells (2.95 to 0.21%). The combination of CD25 depletion andIL-21 treatment resulted in a drop in the CD4+FoxP3+ population to analmost undetectable level (2.95 to 0.017%); further, a substantialdecrease in the fraction of CD4-negative FoxP3+ cells was observed whichwas not seen with either IL-21 treatment or CD25 depletion alone.

IL-21 fails to induce T_(reg) proliferation. To evaluate the possibilitythat the differential effects of IL-21, compared with the othergamma-chain receptor cytokines, IL-2, IL-7 and IL-15, was associatedwith cytokine-mediated T_(reg) proliferation, sorted CD4+25+ T_(reg)cells were treated with IL-2, IL-7, IL-15 or IL-21 in the presence orabsence of CD3-stimulation (FIG. 6A). Anti-CD3 stimulation led toenhanced T_(reg) proliferation (thymidine uptake) among IL-2, IL-7 andIL-15. IL-15 enhanced T_(reg) expansion in a antigen-independent manner.IL-21 however failed to significantly induce T_(reg) proliferation, evenafter anti-CD3 stimulation and was no different from the no cytokineculture.

IL-21 partially reverses T_(reg)-mediated suppression of CD8proliferation. When added to CD8+ T cell cultures, T_(reg) cells inhibitproliferation by up to 66% when evaluated by thymidine incorporationassays (FIG. 6B, * p<0.01 vs. CD8+ only). When IL-21 was added atoptimal concentration, the suppressive effect of T_(reg) on CD8 T cellproliferation was partially reversed. (FIG. 6B, # p<0.01 vs. CD8+plusIL-21). Thus, IL-21 not only fails to induce T_(reg) proliferation, butalso reverses T_(reg) mediated suppression.

C. Discussion

Using a prototypic human self antigen we demonstrate for the first timethat regulatory T cell depletion can lead to substantially enhancedgeneration of tumor-associated antigen-specific CTL. The combination ofCD25 depletion and IL-21 resulted in a >100-fold depletion of FoxP3+T_(reg) cells and augmented the frequency and absolute number oftumor-antigen specific CD8+ CTL between 150 and 300-fold. This effect ismuch greater than that expected given the individual contribution ofeach factor and is unique for IL-21 since the addition of other γ-chainreceptor cytokines such as IL-2, IL-7 and IL-15 did not have a similareffect.

Although CD25 T cell depletion was nearly complete after anti-CD25immunomagnetic depletion, more than 50% of the FoxP3+ populationremained, likely due to the presence of FoxP3+ T_(reg) that do notconstitutively express CD25. IL-21 treatment had a greater FoxP3depleting effect (>90% depletion) and was only able to partially reverseT_(reg)-mediated suppression of CD8 T cell proliferation. Perhaps it wasthe unexpectedly potent combination of both CD25 depletion and IL-21treatment leading to >99.5% depletion of FoxP3+ cells that enabled auniquely robust antigen-specific T cell response. We also observed adecrease of FoxP3+ cells not only in the conventional CD4 compartment,but also among CD8+(CD4-negative) T cells which may represent theputative CD8+ regulatory T cell population recently described.^(27,28)

The mechanism of action of IL-21 in this model is unclear. Uponactivation, both CD4+25+ T_(reg) and CD8+ T cells upregulate IL-21receptor expression but resting T_(reg) express very low levels of IL-21receptor (data not shown and²⁹). Since T_(reg) are not directlyactivated in this model, the decrease in FoxP3+ cells following IL-21treatment in vitro may be due to IL-21 engagement of very low density ofsurface receptors, the indirect action of IL-21 through intermediatecell types, or expansion of effector cells contributing as a cytokinesink for T_(reg) survival.

In recent studies, IL-21 appears to be a key element in the induction ofTh17 cells responsible for proinflammatory and autoimmuneresponses³⁰⁻³². IL-21 deficient T cells failed to differentiate intoTh17 cells and was associated with the reciprocal development of Foxp3Treg cells suggesting a possible role for IL-21 in mediating decreasedlevels of Foxp3 in our findings by enhancing the development of Th17cells at the expense of Treg cells. A genetic basis for these resultsinvolving the idd3 gene which regulates the function of Treg cells wassuggested.³³ These possible mechanisms are currently under investigationin our model.

The observation that IL-21 treatment leads to depletion of FoxP3+ cellscontrasts with results recently published describing the absence of anyT_(reg) depleting effect of IL-21 and no beneficial effect on CD8 T cellproliferation in the presence of T_(reg) ²⁹. Our studies have shown thatthe concentration of IL-21 used may produce confounding results. Atconcentrations greater or equal to 100 ng/ml, there is no significantbeneficial in vitro effect and, in fact, an inhibitory effect. Peluso etal were also unable to address the influence of IL-21 on the generationof antigen-specific CTL since antibodies were used to non-specificallytrigger the T cell receptor to reproduce an ‘inflammatory’ state²⁹. Theuse of anti-CD3 to stimulate through the TCR fails to address issuesassociated with the generation of tumor-associated-, self-antigenspecific T cell responses that arise from a naturally occurring lowfrequency and low avidity T cell subset.

Our recent experience in human samples demonstrated a beneficial effectof IL-21 on the generation of helper-independent antigen-specific CTL²³.The impact of IL-21 on T_(reg) cells has only recently been proposedand, until now, not previously shown to directly or indirectly depletehuman T_(reg) population. Neither the means to eliminate FoxP3+ cells tosuch a profound degree nor the capacity to enhance the generation oftumor-associated antigen-specific CTL has previously been demonstrated.By removing regulatory constraints limiting antigen-specific expansion,this strategy may not only improve the prospects for generatingtumor-reactive T cells for adoptive cellular therapy, but also uncovernovel, tumor-associated targets that were previously below the thresholdfor generating in vitro T cell responses.

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The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A method of making a cytotoxic T lymphocyte (CTL) preparation useful for adoptive immunotherapy, comprising: (a) sorting a lymphocyte subpopulation depleted of CD25⁺ cells from a first lymphocyte population of peripheral blood mononuclear cells (PBMC) to produce a CD8⁺CD25⁻subpopulation; (b) promoting the production of antigen-specific CTL cells in said subpopulation by in vitro culturing the sorted CD8⁺CD25⁻ subpopulation with antigen in a medium containing interleukin-21; and (c) formulating antigen-specific CTL cells produced therefrom into a pharmaceutical formulation.
 2. The method of claim 1, wherein said interleukin-21 is included in said culture in an amount of from 1 to 1000 nanograms per milliliter.
 3. The method of claim 1, wherein the CTL cells are specific for a tumor antigen.
 4. The method of claim 1, wherein (a) the sorting a lymphocyte subpopulation depleted of CD25⁺ cells from a first lymphocyte population comprises treating the first lymphocyte population with denileukin diftitox.
 5. The method of claim 1, wherein (a) the sorting a lymphocyte subpopulation depleted of CD25⁺ cells from a first lymphocyte population comprises contacting the first lymphocyte population with anti-CD25 antibodies.
 6. The method of claim 1, wherein (b) the promoting the production of antigen-specific CTL cells in said subpopulation by in vitro culturing comprises culturing the CTLs with antigen presenting cells.
 7. The method of claim 1, wherein the antigen presenting cells comprise dendritic cells (DCs).
 8. The method of claim 1, wherein the antigen-specific CTL cells comprise a transgene.
 9. The method of claim 1, wherein the antigen-specific CTL cells of the formulation consist essentially of CD8⁺CD25− antigen-specific CTL cells.
 10. The method of claim 1, wherein the number of antigen specific CTL are enriched in said CD8⁺CD25⁻ subpopulation by at least 100-fold, as compared to that seen in the same lymphocyte population not subjected to either the sorting step (a) or the promoting step (b).
 11. The method of claim 1, wherein the pharmaceutical formulation comprises at least 10⁹ CTL cells. 